Quantitating human risk from radiation has been a controversial area for many years, in large part due to the lack of a detailed understanding of the molecular nature of radiation damage to DNA and other potentially important cellular molecules. Studies of acute vs. split dose radiation induced malignant transformation of cultured mammalian cells, as well as of enhancement of in vivo chemical carcinogenesis, have in several instances suggested that at total doses of less than 200 R, split doses may be more effective at inducing malignant transformation and at enhancing chemical carcinogenesis than are acute exposures of the same total dose. We propose to develop shuttle vector techniques to study the specific DNA damage caused by acute vs. split dose gamma radiation across a wide radiation dose range. Background mutation rates will be established for extrachromosomal and retroviral shuttle vectors, both carrying the same inserted DNA sequence (lacI). Baseline studies will address the mutagenic effects of exposures to various acute doses of gamma-radiation on the DNA insert. Resulting DNA damage will be analyzed by genetic techniques and/or by recombinant DNA techniques subsequent to reisolation of shuttle DNA from E. coli. This system will be subjected to either acute gamma-ray doses or 2 half-sized gamma-ray doses separated by 1,5 or 24-hour intervals at total doses ranging from 0.01-100.0Gy. Radiation will be administered to either the intact shuttle prior to transfection into the human 293 cell, or the shuttle-containing human 293 cell. Additionally some groups of 293 cells will be irradiated prior to insertion of irradiated or non-irradiated shuttles. Shuttle inserts will be analyzed for specific DNA lesions, including deletions and point mutations. This damage will be related to the extrachromosomal vs. intra-host genomic nature of the shuttle, acute vs. split radiation exposures, and the timing of radiation exposures. Such protocols will also allow evaluation of the role of the human host cell on the repair of radiation damage to the shuttle. This model should provide an excellent means of studying specific molecular events involved in radiation induced DNA damage and repair, as well as of further exploring the differences between DNA damage induced by acute and split doses of radiation at low total doses.